Studies over the last funding period were directed to elucidating the molecular basis for transport-associated acquired resistance to antifolates in murine leukemia cells and gaining insight into the structure-function of the murine RFC (RFC). The current proposal represents an evolution of this work, based on recent studies from this laboratory, that have characterized ubiquitously expressed, prominent, folate transport activities with low-pH optima in human solid tumor cell lines that contribute to the activity of antifolates and play a role in intestinal absorption of folates. The data indicate that there are at least two types of low-pH folate transport activities typified by (i) rat intestinal epithelial cells, (IEC-6) in which the major portion, but not all, of the low pH activity is linked in some way to RFC expression and (ii) Hela cells in which the low-pH activity is entirely RFC- independent. The objectives of the proposed studies are to explore mechanisms underlying transport-related antifolate resistance in human solid tumor cell lines and to better define the basis for, and pharmacological importance of, low-pH folate transport routes and their relationship to RFC. The new generation antifolate, pemetrexed, is emphasized in these studies because of increasing evidence of its clinical utility, its apparent high affinity for low pH transporter(s), and its other novel properties. The studies proposed: (i) assess the prevalence, and characterize the properties of, folate transport activities with low-pH optima; (ii) develop RFC-deficient human solid tumor cell lines in order to distinguish between RFC-associated, and RFC-independent, low pH transport activities, to provide further insight into the structure-function of human RFC, and how this impacts on antifolate activities; (iii) select human tumor cell lines for primary resistance to pemetrexed in order to assess the roles of RFC and/or low-pH transport activities, assess cross-resistance patterns, and determine the role of putative pemetrexed enzyme targets in drug activity; (iv) clarify mechanisms by which RFC function is related to low-pH transport activity in cells of small intestinal origin, and possibly tumors that may be identified; (v) implement methodologies for cloning the low pH. RFC-independent, transporter in Hela cells. The proposed studies are supported by preliminary data and variety of cell lines that have been developed with impaired RFC function, altered low pH-transport activity, or other unique properties that will facilitate this work.